Matilda F. Chan

Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements

ABSTRACT We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.

Reduced Rates of Gene Loss, Gene Silencing, and Gene Mutation in Dnmt1-Deficient Embryonic Stem Cells

ABSTRACT Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by cancer type. The factors that influence the relative utilization of gene inactivation pathways are poorly understood. In this study, we describe a detailed quantitative analysis of the three major gene inactivation mechanisms for a model gene at two different genomic integration sites in mouse embryonic stem (ES) cells. In addition, we targeted the major DNA methyltransferase gene, Dnmt1, to investigate the relative contribution of DNA methylation to these various competing gene inactivation pathways. Our data show that gene loss is the predominant mode of inactivation of a herpes simplex virus thymidine kinase neomycin phosphotransferase reporter gene (HSV-TKNeo) at the two integration sites tested and that this event is significantly reduced in Dnmt1-deficient cells. Gene silencing by promoter methylation requires Dnmt1, suggesting that the expression of Dnmt3a and Dnmt3b alone in ES cells is insufficient to achieve effective gene silencing. We used a novel assay to show that missense mutation rates are also substantially reduced in Dnmt1-deficient cells. This is the first direct demonstration that DNA methylation affects point mutation rates in mammalian cells. Surprisingly, the fraction of CpG transition mutations was not reduced in Dnmt1-deficient cells. Finally, we show that methyl group-deficient growth conditions do not cause an increase in missense mutation rates in Dnmt1-proficient cells, as predicted by methyltransferase-mediated mutagenesis models. We conclude that Dnmt1 deficiency and the accompanying genomic DNA hypomethylation result in a reduction of three major pathways of gene inactivation in our model system.

The Clinical Differentiation of Bacterial and Fungal Keratitis: A Photographic Survey

PURPOSE The purpose of this study was to determine whether clinical signs of infectious keratitis can be used to identify the causative organism. METHODS Eighty photographs of eyes with culture-proven bacterial keratitis or smear-proven fungal keratitis were randomly selected from 2 clinical trials. Fifteen cornea specialists from the F. I. Proctor Foundation and the Aravind Eye Care System assessed the photographs for prespecified clinical signs of keratitis, and they identified the most likely causative organism. RESULTS Clinicians were able to correctly distinguish bacterial from fungal etiology 66% of the time (P < 0.001). The Gram stain, genus, and species were accurately predicted 46%, 25%, and 10% of the time, respectively. The presence of an irregular/feathery border was associated with fungal keratitis, whereas a wreath infiltrate or an epithelial plaque was associated with bacterial keratitis. CONCLUSIONS Cornea specialists correctly differentiated bacterial from fungal keratitis more often than chance, but in fewer than 70% of cases. More specific categorization led to less successful clinical distinction. Although certain clinical signs of infectious keratitis may be associated with a bacterial or fungal etiology, this study highlights the importance of obtaining appropriate microbiological testing during the initial clinical encounter.

The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair

Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs) are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS). SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1−/−, but not Sulf2−/−, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1−/− mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE). Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

Protective effects of matrix metalloproteinase-12 following corneal injury

Summary Corneal scarring due to injury is a leading cause of blindness worldwide and results from dysregulated inflammation and angiogenesis during wound healing. Here we demonstrate that the extracellular matrix metalloproteinase MMP12 (macrophage metalloelastase) is an important regulator of these repair processes. Chemical injury resulted in higher expression of the fibrotic markers &agr;-smooth muscle actin and type I collagen, and increased levels of angiogenesis in corneas of Mmp12−/− mice compared with corneas of wild-type mice. In vivo, we observed altered immune cell dynamics in Mmp12−/− corneas by confocal imaging. We determined that the altered dynamics were the result of an altered inflammatory response, with delayed neutrophil infiltration during the first day and excessive macrophage infiltration 6 days later, mediated by altered expression levels of chemokines CXCL1 and CCL2, respectively. Corneal repair returned to normal upon inhibition of these chemokines. Taken together, these data show that MMP12 has a protective effect on corneal fibrosis during wound repair through regulation of immune cell infiltration and angiogenesis.

Late varicella-zoster virus dendriform keratitis in patients with histories of herpes zoster ophthalmicus.

PURPOSE To describe the characteristics and course of late varicella-zoster virus (VZV) dendriform keratitis in patients with histories of herpes zoster ophthalmicus (HZO); to describe responses of corneal lesions to antiviral treatment; and to investigate risk factors for recurrence. DESIGN Retrospective case series. METHODS Included were patients known to have 1 or more episodes of dendriform lesions beginning at least 2 weeks after HZO in 2 academic practices. Epithelial lesions were evaluated for the presence of VZV DNA by a polymerase chain reaction assay. Demographic, medical, and ophthalmic data were collected for each episode. Responses to treatment with antiviral medications were evaluated. Cumulative risk of recurrence was determined using Kaplan-Meier analysis; potential risk factors for recurrence (age, systemic disease, lesion characteristics, corticosteroids) were evaluated using univariate Cox proportional hazard models. RESULTS We identified 20 patients (14 women; median age, 65 years) who met inclusion criteria. Dendriform lesions were pleomorphic with thickened, opaque epithelium. Seven patients had systemic diseases characterized by altered immune function. VZV DNA was identified in 15 of 16 cases tested, and all lesions responded to antiviral therapy. The 1-year incidence of first recurrence was 95.8 lesions per 100 person-years of follow-up. Patients had multiple recurrences, but risk of recurrence appeared to decrease over time. No statistically significant risk factors for recurrence were identified. CONCLUSIONS Late dendriform lesions associated with HZO are foci of productive VZV infection. Lesions can be treated effectively with topical or systemic antiviral agents. Patients can have multiple recurrences of dendriform lesions despite treatment.

Membrane array analysis of tear proteins in ocular cicatricial pemphigoid.

Purpose. To explore non-invasive, protein-based, membrane array technology as a means to evaluate the global immune and angiogenic profile of tear proteins in patients with active ocular cicatricial pemphigoid (OCP). Methods. Forty-three proteins consisting of cytokines, angiogenic/growth factors, and immunoinflammatory modulators were measured by membrane array in tear samples of four control patients and four OCP patients during active disease and after treatment. Results. Signals for several distinct and consistent molecular entities were upregulated in all four active OCP tear samples relative to controls. In particular, interleukin-8 and matrix metalloproteinase-9 were elevated during active disease and decreased after systemic immunomodulatory therapy. Conclusions. Protein array analysis may provide a well-tolerated assay to monitor levels of inflammatory markers in the tears of OCP patients in response to therapy.

Microbiological cure times in acanthamoeba keratitis.

AimsThe purpose of this study was to estimate the duration of treatment necessary for sequential acanthamoeba laboratory tests from corneal scrapings to become negative, and to assess predictors that affect this duration period.MethodsWe included all patients with at least one positive acanthamoeba culture or Giemsa stain at the F.I. Proctor Foundation Microbiology Laboratory from 1996 to 2009. A parametric survival analysis was performed among patients with repeat cultures to assess significant predictors for extended clearance time. Simulations were performed to estimate clearance time in the entire patient population, assuming imperfect sensitivity.ResultsThirty-seven patients with laboratory evidence of acanthamoeba had testing at 69 time points. The median clearance time among eyes with repeat cultures was 42.5 days (interquartile range (IQR) 22.0–82.0 days; unadjusted parametric model). Initial visual acuity was the only predictor significantly associated with clearance time in univariate analyses (P<0.0001). Using initial visual acuity as a predictor for clearance time among the entire patient population, the estimated clearance time decreased to 38.7 days (95% confidence interval (CI) 27.9–53.5 days). When the imperfect sensitivity of the culture technique was also taken into account, the estimated clearance time was 44.1 days (95% CI 31.9–61.0 days).ConclusionThe duration of infection with acanthamoeba keratitis undergoing treatment has not been well characterized. In this report we estimate a median clearance time of approximately 6 weeks, with an IQR of 22–82 days.

Metalloproteinases: A functional pathway for myeloid cells

Myeloid cells have diverse roles in regulating immunity, inflammation, and extracellular matrix turnover. To accomplish these tasks, myeloid cells carry an arsenal of metalloproteinases, which include the matrix metalloproteinases and the adamalysins. These enzymes have diverse substrate repertoires, and are thus involved in mediating proteolytic cascades, cell migration, and cell signaling. Dysregulation of metalloproteinases contributes to pathogenic processes, including inflammation, fibrosis, and cancer. Metalloproteinases also have important nonproteolytic functions in controlling cytoskeletal dynamics during macrophage fusion and enhancing transcription to promote antiviral immunity. This review highlights the diverse contributions of metalloproteinases to myeloid cell functions.

Cicatrising conjunctivitis due to paraneoplastic pemphigoid

Ocular cicatricial pemphigoid (OCP) is a putative autoimmune, chronic, inflammatory, subepithelial blistering disease affecting mucous membranes.1 It is characterised by linear deposition of immunoglobulins and complement along epithelial basement membranes.1 In this report, we describe a cicatrising conjunctivitis that was clinically and pathologically similar to ocular cicatricial pemphigoid but occurred concurrently with systemic malignancy. A 69-year-old cachectic man with chronic obstructive pulmonary disease presented with painful mouth and skin blisters. He developed a painful red eye 1 week later. Visual acuity was 20/25 on the right and 20/30 on the left. Slit-lamp examination showed bulbar conjunctival injection and an intense papillary reaction of the palpebral conjunctiva on …